The research interests of this laboratory are centered on the molecular details of animal RNA virus replication and their interactions with vertebrate and invertebrate hosts. The experiments detailed in this proposal concern studies on yellow fever virus (YF), type virus of the family Flaviviridae. Several members of this family are agents of arthropod-transmitted world health problems, and our long range goals include a thorough understanding of viral RNA replication and gene expression, virion assembly, and viral pathogenesis and host immunity. Although several flavivirus genome sequences are known, the functions of the viral proteins and conserved RNA sequences/structures in replication are poorly understood. This proposal focuses on a biochemical and molecular genetic dissection of the flavivirus proteinase, its cleavage sites in the viral polyprotein, and the effects cleavage site and proteinase mutations on YF replication. It is likely that a trypsin-like serine proteinase domain, localized in the N-terminal one third the nonstructural protein, NS3, mediates five cleavages essential for at least two key steps in productive virus infection: 1) formation of the RNA replication complex and 2) assembly of the virion. The proteinase and its cleavage sties represent well-defined targets for mutagenesis studies which should yield insight into both these processes. The YF system is unique among the Flaviviridae allowing study of the effects of such mutations using both in vitro and in vivo assays for cis and trans cleavage and also analysis of their effects on YF RNA replication and virus formation, given the ability to generate infectious YF RNA from cDNA. Preliminary studies indicate that a second nonstructural protein, NS2B, perhaps complexed with NS3, may also be required for efficient site-specific proteolysis. In vitro assays for trans cleavage activity will facilitate purification of active proteinases for determination of subunit composition and substrate specificity, for screening potential proteinase inhibitors, and for high resolution structural analyses. The information gained from such studies should further our understanding of serine proteinase structure and function and aid in the design of flavivirus-specific proteinase inhibitors as potential antiviral therapy.